Abstract
Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.
Original language | English (US) |
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Pages (from-to) | 3192-3208.e11 |
Journal | Molecular cell |
Volume | 84 |
Issue number | 17 |
DOIs | |
State | Published - Sep 5 2024 |
Funding
The authors would like to thank Anita Wang for assistance with reagent preparations and experiment coordination and Steve Lauberth for graphic design and figure layout preparation. All mass spectrometry was performed by the Biomolecular and Proteomics Mass Spectrometry Facility, UC San Diego (Majid Ghassemian, PhD, Director). All BLI experiments were performed with the help of the Northwestern University Keck Biophysics Facility. Support from the R.H. Lurie Comprehensive Cancer Center of Northwestern University to the Keck Biophysics Facilities is acknowledged. Research in the Lauberth lab is supported by a grant from the NIH/National Institute of General Medical Sciences (R35 GM128900) to S.M.L. Cancer Center research in the Mondrag\u00F3n Lab is supported by the NIH (R35-GM118108) to A.M. and an NIH NRSA (5T32-GM008382) training grant fellowship to E.T. Research in the John Marko Lab is supported by the NIH (R01-GM105847) to J.F.M. E.L.V.N. is a CPRIT Scholar in Cancer Research (RR200040), and research in the Chuan lab is supported by the following funding: C.H. RM1 HG008935. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program (ET) under Grant No. (DGE-2234667). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. The research reported in this publication was supported by NIGMS of the National Institutes of Health under award number T32GM140995 (E.T.). Conceptualization, S.M.L. P.O. H.R. M.B. and K.A.; methodology, most of the experiments were performed by P.O. H.R. K.A. and S.M.L.; computational analysis, H.R. P.O. M.B. K.A. S.M.L. and B.L.; software, M.B.; investigation, P.O. H.R. M.B. K.A. E.T. N.M. B.L. P.A.-L. P.P. J.F.M. S.M.L. and A.C.S.; resources, J.F.M. S.M.L. E.L.V.N. and A.M.; writing \u2013 review and editing, S.M.L. E.T. P.O. H.R. A.M. C.H. M.B. K.A. and E.L.V.N.; funding acquisition, S.M.L. All authors critically reviewed the manuscript and approved the final version. E.L.V.N. is co-founder, member of the Board of Directors, on the SAB, equity holder, and paid consultant for Eclipse BioInnovations. E.L.V.N.\u2019s interests have been reviewed and approved by the Baylor College of Medicine in accordance with its conflict-of-interest policies. C.H. is a scientific founder, a member of the scientific advisory board and equity holder of Aferna Bio, Inc. and Ellis Bio Inc. a scientific co-founder and equity holder of Accent Therapeutics, Inc. and a member of the scientific advisory board of Rona Therapeutics and Element Biosciences. The authors would like to thank Anita Wang for assistance with reagent preparations and experiment coordination and Steve Lauberth for graphic design and figure layout preparation. All mass spectrometry was performed by the Biomolecular and Proteomics Mass Spectrometry Facility, UC San Diego (Majid Ghassemian, PhD, Director). All BLI experiments were performed with the help of the Northwestern University Keck Biophysics Facility. Support from the R.H. Lurie Comprehensive Cancer Center of Northwestern University to the Keck Biophysics Facilities is edged. Research in the Lauberth lab is supported by a grant from the NIH/National Institute of General Medical Sciences ( R35 GM128900 ) to S.M.L. Cancer Center research in the Mondrag\u00F3n Lab is supported by the NIH ( R35-GM118108 ) to A.M. and an NIH NRSA ( 5T32-GM008382 ) training grant fellowship to E.T. Research in the John Marko Lab is supported by the NIH ( R01-GM105847 ) to J.F.M. E.L.V.N. is a CPRIT Scholar in Cancer Research ( RR200040 ), and research in the Chuan lab is supported by the following funding: C.H. RM1 HG008935 . This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program (ET) under Grant No. ( DGE-2234667 ). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. The research reported in this publication was supported by NIGMS of the National Institutes of Health under award number T32GM140995 (E.T.).
Keywords
- DNA supercoiling
- RNA-binding protein
- TOP1
- TOP1 interactome
- magnetic tweezers
- topology
- transcription
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology